Understanding the mechanisms underlying chronic pain is important to improving clinical treatment and developing novel therapeutic strategies. The AMPA receptor GluR2 subunit, through its binding to GRIP and PICK1, may function in the synaptic AMPA receptor trafficking that is critical for many forms of neuronal plasticity. GRIP anchors GluR2 at synapses, whereas PICK1 brings PKC1 to synaptic GluR2. PKC1 phosphorylates GluR2 Ser880 to release GluR2 from GRIP and to promote GluR2 internalization in vitro. We have made the novel discoveries that Complete Freund's adjuvant (CFA)-induced peripheral inflammation and spinal nerve injury (SNI) led to the disruption of interaction of spinal GluR2 with GRIP and that activation of spinal NMDA receptors produced an increase in the amount of GluR2 in the 150,000-g spin fraction that contains endocytosed clathrin-coated vesicles, suggesting that CFA-induced inflammatory and SNI-induced neuropathic insults might induce PKC phosphorylation of GluR2 Ser880 and drive GluR2 internalization via NMDA receptor activation in dorsal horn in vivo. This proposal seeks to determine whether and how GluR2 is internalized in dorsal horn neurons under chronic pain conditions and whether this internalization contributes to the central sensitization underlying chronic pain. In Specific Aim 1, we will determine (a) if CFA injection and SNI time-dependently increase the level of GluR2 phosphorylation at Ser880 and the amount of GluR2 protein in 150,000- g spin fraction derived from dorsal horn, (b) if they decrease the surface and synaptic expression of GluR2 in dorsal horn neurons, and (c) if they result in a switch of Ca2+impermeable (GluR2-containing) AMPA receptors expressed on many dorsal horn neurons to Ca2+permeable (GluR2-lacking) AMPA receptors. In Specific Aim 2, we will determine if spinal PKC1 or NMDA receptor activation increases the level of GluR2 phosphorylation at Ser880 and the amount of GluR2 in the 150,000-g fraction in in vitro dorsal horn slices. Furthermore, we will examine if inhibition of spinal PKC1 or NMDA receptor activation attenuates CFA- or SNI-induced increases in the level of GluR2 phosphorylation at Ser880 and the amount of GluR2 in the 150,000-g fraction in in vivo dorsal horn. In Specific Aim 3, we will determine if blocking GluR2 internalization through inhibition of spinal PKC1 activation, blockade of PKC1 recruitment to GluR2 by PICK1 deletion, or targeted mutation of the GluR2 PKC1 phosphorylation site attenuates CFA- or SNI-induced thermal and mechanical pain hypersensitivity. The proposed studies will provide new insights into the mechanisms of both AMPA receptor and NMDA receptor actions on chronic pain and open a door for the development of new analgesics in treatment or prevention of chronic pain.